This program project addresses five aspects of the development of the human erythroid cell. Project I involves studies of the maturation of human and simian erythroid progenitors and includes evaluation of the control of the F to A switch and A to F switches at the progenitor and at differentiated precursor cell levels. In Project II attempts will be made to purify erythropoietin. This reagent will be used to create antibodies to the hormone, document erythropoietin receptors on erythroid progenitors, study crythroid progenitor dysfunctions and purify the progenitors themselves. Project III is devoted to evaluations of certain molecular aspects of gene switching. These include quantitation of the relative outputs of alpha globin mRNA, as well as in states of abnormal erythropoiesis. The project also includes examinations of nuclease sensitive sites and the pattern of methylation in the epsilon-gamma-beta and zeta-alpha globin DNA regions during fetal and adult development. An attempt will be made to construct recombinant plasmids containing erythropoietin cDNA to synthesize erythropoietin in E. coli and develop the complete DNA sequence of the hormone. Project IV focuses on preparation of specific antisera to membrane proteins such as spectrin, ankyrin, actin, glycophorin and proteins 3 and 4.1, determinations of the content and distribution of each of these membrane skeletal proteins in intact cells, skeletons of human bone marrow erythroblasts at different stages of maturation, and in cultured human erythroblasts including K562 cells and erythroblasts derived from CFU-E and mature and immature BFU-E. The project also explores the possibility that erythroid specific proteins such as spectrin, glycophorin A, or protein 4.1 may be present on committed erythroid progenitors. Finally, a comparison will be made of the structural and functional properties of the membrane skeletal proteins of fetal and adult erythrocytes. Project V is devoted to analysis of the synthesis and maturation of major erythrocyte membrane proteins, specifically band 3, spectrin and the spectrin binding proteins 2.1 and 4.1 with particular emphasis on band 3. Studies of in vitro synthesis, glycosylation and membrane insertion of these proteins will be performed.